Summary Telomeres are repetitive DNA and associated proteins at the ends of the chromosomes that are important for genome stability. The shelterin complex, a six-member group of proteins, is important for telomere length maintenance, which occurs through regulating the recruitment and action of the telomerase enzyme. In addition, shelterin also protects the chromosome ends from being incorrectly recognized by DNA damage repair mechanisms. Disorders in telomere biology are an underlying cause of many human diseases. Mutation of telomerase or shelterin components, resulting in loss of telomere maintenance, leads to dyskeratosis congenita, pulmonary fibrosis and aplastic anemia. Its medical importance is further shown by the fact that 90% of cancers depend on hyper-activation of telomerase for persistent proliferation. Despite the critical roles shelterin plays in genome stability, a consistent framework of understanding its mechanism in telomere maintenance has not been established. A leading model of how shelterin can protect chromosome ends and restrict telomerase access to the telomeric tail is the telomere-loop model. Other simpler models include DNA end-capping or compaction by shelterin to form reclusive structures. While these models are derived from the DNA remodeling properties of individual shelterin proteins, the collective roles shelterin proteins play as a functional multisubunit complex are still ambiguous. How a shelterin complex organizes telomeric DNA into various architectures for their regulatory functions and how they switch between inhibitory and permissive roles in telomerase elongation of telomeres are the next key questions. The answers lie in the molecular mechanisms of these processes and hence, the goal of this proposal is to elucidate the structural basis of shelterin complex functions in regulating telomerase recruitment and elongation of telomeres. A multiscale and interdisciplinary approach will be used, which includes biochemistry, biophysics, cell biology and cryo-EM techniques. Specifically, this proposal aims to determine: (1) How the shelterin complex organizes telomeric DNA into various architectures that regulate telomerase accessibility, (2) The structure of the shelterin complex assembled with telomeric DNA, and (3) How a shelterin complex switches from telomere end-capping to telomerase stimulation. During the K99 phase, under the mentorship of Dr. Tom Cech, AFM imaging with biochemical assays will be used to characterize higher-order DNA-protein architectures formed by various shelterin complexes, and their effects on telomerase recruitment and activity will be measured. With additional support from Dr. Zhiheng Yu, a cryo-EM skill set will be acquired while determining the cryo-EM structure of shelterin in association with telomeric DNA. This will facilitate using cryo-EM to study the structural basis of telomerase regulation by shelterin during the independent R00 phase. The results of this proposal would provide a multiscale framework for understanding the mechanisms of shelterin functions in telomere maintenance, and also potentially provide new avenues in developing therapeutic and diagnostic strategies to combat human diseases of telomere dysfunction.